Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity

ABSTRACT

The present invention relates to the field of glycosylation engineering of proteins. More particularly, the present invention relates to glycosylation engineering to generate proteins with improved therapeutic properties, including antibodies with increased antibody-dependent cellular cytotoxicity.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/665,191 filed Mar. 23, 2015, now allowed. U.S. patent applicationSer. No. 14/665,191 is a divisional of U.S. patent application Ser. No.13/196,724 filed Aug. 2, 2011, now U.S. Pat. No. 8,999,324, issued Apr.7, 2015. U.S. patent application Ser. No. 13/196,724 is a continuationof U.S. patent application Ser. No. 11/199,232, filed Aug. 9, 2005, nowU.S. Pat. No. 8,021,856, issued Sep. 20, 2011. U.S. patent applicationSer. No. 11/199,232 is a continuation of U.S. application Ser. No.10/211,554, now abandoned, filed Aug. 5, 2002, which claims the benefitof U.S. Provisional App. No. 60/309,516, filed Aug. 3, 2011, and acontinuation-in-part of U.S. application Ser. No. 10/633,697, filed Aug.5, 2003, now U.S. Pat. No. 7,517,670, issued Apr. 14, 2009, which is adivisional of U.S. patent application Ser. No. 09/294,584, filed on Apr.20, 1999, now U.S. Pat. No. 6,602,684, which claims the benefit of U.S.Provisional App. No. 60/082,581, filed Apr. 20, 1998. These applicationsare incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of glycosylation engineeringof proteins. More particularly, the present invention relates toglycosylation engineering to generate proteins with improved therapeuticproperties, including antibodies with increased antibody-dependentcellular cytotoxicity.

2. Background Art

Glycoproteins mediate many essential functions in human beings, othereukaryotic organisms, and some prokaryotes, including catalysis,signaling, cell-cell communication, and molecular recognition andassociation. They make up the majority of non-cytosolic proteins ineukaryotic organisms. (Lis et al., Eur. J. Biochem. 218:1-27 (1993)).Many glycoproteins have been exploited for therapeutic purposes, andduring the last two decades, recombinant versions ofnaturally-occurring, secreted glycoproteins have been a major product ofthe biotechnology industry. Examples include erythropoietin (EPO),therapeutic monoclonal antibodies (therapeutic mAbs), tissue plasminogenactivator (tPA), interferon-β, (IFN-β), granulocyte-macrophage colonystimulating factor (GM-CSF), and human chorionic gonadotrophin (hCG).(Cumming et al., Glycobiology 1:115-130 (1991)).

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins et al.,Nature Biotechnol. 14:975-81 (1996)).

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. (Cumming et al.,Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol.14:975-81 (1996)). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the blood stream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum-free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NS0- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins et al., Nature Biotechnol.14:975-81 (1996)).

All antibodies contain carbohydrate structures at conserved positions inthe heavy chain constant regions, with each isotype possessing adistinct array of N-linked carbohydrate structures, which variablyaffect protein assembly, secretion or functional activity. (Wright, A.,and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). The structure ofthe attached N-linked carbohydrate varies considerably, depending on thedegree of processing, and can include high-mannose, multiply-branched aswell as biantennary complex oligosaccharides. (Wright, A., and Morrison,S. L., Trends Biotech. 15:26-32 (1997)). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a particular glycosylation site such that even monoclonal antibodiesexist as multiple glycoforms. Likewise, it has been shown that majordifferences in antibody glycosylation occur between cell lines, and evenminor differences are seen for a given cell line grown under differentculture conditions. (Lifely, M. R. et al., Glycobiology 5(8):813-22(1995)).

Unconjugated monoclonal antibodies (mAbs) can be useful medicines forthe treatment of cancer, as demonstrated by the U.S. Food and DrugAdministration's approval of Rituximab (Rituxan™; IDEC Pharmaceuticals,San Diego, Calif., and Genentech Inc., San Francisco, Calif.), for thetreatment of CD20 positive B-cell, low-grade or follicular Non-Hodgkin'slymphoma, and Trastuzumab (Herceptin™; Genentech Inc) for the treatmentof advanced breast cancer (Grillo-Lopez, A.-J., et al., Semin. Oncol.26:66-73 (1999); Goldenberg, M. M., Clin. Ther. 21:309-18 (1999)). Thesuccess of these products relies not only on their efficacy but also ontheir outstanding safety profiles (Grillo-Lopez, A.-J., et al., Semin.Oncol. 26:66-73 (1999); Goldenberg, M. M., Clin. Ther. 21:309-18(1999)). In spite of the achievements of these two drugs, there iscurrently a large interest in obtaining higher specific antibodyactivity than what is typically afforded by unconjugated mAb therapy.

One way to obtain large increases in potency, while maintaining a simpleproduction process and potentially avoiding significant, undesirableside effects, is to enhance the natural, cell-mediated effectorfunctions of mAbs by engineering their oligosaccharide component (Umaña,P. et al., Nature Biotechnol. 17:176-180 (1999)). IgG1 type antibodies,the most commonly used antibodies in cancer immunotherapy, areglycoproteins that have a conserved N-linked glycosylation site atAsn297 in each CH2 domain. The two complex biantennary oligosaccharidesattached to Asn297 are buried between the CH2 domains, forming extensivecontacts with the polypeptide backbone, and their presence is essentialfor the antibody to mediate effector functions such as antibodydependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al.,Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol Rev.163:59-76 (1998); Wright, A. and Morrison, S. L., Trends Biotechnol.15:26-32 (1997)).

The present inventors showed previously that overexpression in Chinesehamster ovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III(GnTIII), a glycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umaña, P. et al., Nature Biotechnol.17:176-180 (1999), International Publication No. WO 99/54342, the entirecontents of each of which are hereby incorporated by reference in theirentirety). The antibody chCE7 belongs to a large class of unconjugatedmAbs which have high tumor affinity and specificity, but have too littlepotency to be clinically useful when produced in standard industrialcell lines lacking the GnTIII enzyme (Umana, P., et al., NatureBiotechnol. 17:176-180 (1999)). That study was the first to show thatlarge increases of maximal in vitro ADCC activity could be obtained byincreasing the proportion of constant region (Fc)-associated, bisectedoligosaccharides above the levels found in naturally occurringantibodies. To determine if this finding could be extrapolated to anunconjugated mAb, which already has significant ADCC activity in theabsence of bisected oligosaccharides, the present inventors have appliedthis technology to Rituximab, the anti-CD20, IDEC-C2B8 chimericantibody. The present inventors have likewise applied the technology tothe unconjugated anti-cancer mAb chG250.

BRIEF SUMMARY OF THE INVENTION

The present inventors have now generated new glycosylation variants ofthe anti-CD20 monoclonal antibody (mAb) IDEC-C2B8 (Rituximab) and theanti-cancer mAb chG250 using genetically engineered mAb-producing celllines that overexpress N-acetylglucosaminyltransferase III (GnTIII; EC2.1.4.144) in a tetracycline regulated fashion. GnTIII is required forthe synthesis of bisected oligosaccharides, which are found at low tointermediate levels in naturally-occurring human antibodies but aremissing in mAbs produced in standard industrial cell lines. The newglycosylated versions outperformed Mabthera™ (the version of Rixtuximabmarketed in Europe) and mouse-myeloma derived chG250 in biological(ADCC) activity. For example, a ten-fold lower amount of the variantcarrying the highest levels of bisected oligosaccharides was required toreach the maximal ADCC activity as Mabthera™. For chG250, the variantcarrying the highest levels of bisected oligosaccharides mediatedsignificant ADCC activity at a 125-fold lower concentration than thatrequired to detect even low ADCC activity by the unmodified controlchG250. A clear correlation was found between the level of GnTIIIexpression and ADCC activity.

Accordingly, in one aspect the claimed invention is directed to a hostcell engineered to produce a polypeptide having increased Fc-mediatedcellular cytotoxicity by expression of at least one nucleic acidencoding β(1,4)-N-acetylglucosaminyltransferase III (GnT III), whereinthe polypeptide produced by the host cell is selected from the groupconsisting of a whole antibody molecule, an antibody fragment, and afusion protein which includes a region equivalent to the Fc region of animmunoglobulin, and wherein the GnT III is expressed in an amountsufficient to increase the proportion of said polypeptide carryingbisected hybrid oligosaccharides or galactosylated complexoligosaccharides or mixtures thereof in the Fc region relative topolypeptides carrying bisected complex oligosaccharides in the Fcregion.

In a preferred embodiment, the polypeptide is IgG or a fragment thereof,most preferably, IgG1 or a fragment thereof. In a further preferredembodiment, the polypeptide is a fusion protein that includes a regionequivalent to the Fc region of a human IgG.

In another aspect of the claimed invention, a nucleic acid moleculecomprising at least one gene encoding GnTIII has been introduced intothe host cell. In a preferred embodiment, at least one gene encodingGnTIII has been introduced into the host cell chromosome.

Alternatively, the host cell has been engineered such that an endogenousGnT III gene is activated, for example, by insertion of a DNA elementwhich increases gene expression into the host chromosome. In a preferredembodiment, the endogenous GnTIII has been activated by insertion of apromoter, an enhancer, a transcription factor binding site, atransposon, or a retroviral element or combinations thereof into thehost cell chromosome. In another aspect, the host cell has been selectedto carry a mutation triggering expression of an endogenous GnTIII.Preferably, the host cell is the CHO cell mutant lec 10.

In a further preferred embodiment of the claimed invention, the at leastone nucleic acid encoding a GnTIII is operably linked to a constitutivepromoter element.

In a further preferred embodiment, the host cell is a CHO cell, a BHKcell, a NS0 cell, a SP2/0 cell, or a hybridoma cell, a Y0 myeloma cell,a P3X63 mouse myeloma cell, a PER cell or a PER.C6 cell and saidpolypeptide is an anti-CD20 antibody. In another preferred embodiment,the host cell is a SP2/0 cell and the polypeptide is the monoclonalantibody chG250.

In another aspect, the claimed invention is directed to a host cell thatfurther comprises at least one transfected nucleic acid encoding anantibody molecule, an antibody fragment, or a fusion protein thatincludes a region equivalent to the Fc region of an immunoglobulin. In apreferred embodiment, the host cell comprises at least one transfectednucleic acid encoding an anti-CD20 antibody, the chimeric anti-humanneuroblastoma monoclonal antibody chCE7, the chimeric anti-human renalcell carcinoma monoclonal antibody chG250, the chimeric anti-humancolon, lung, and breast carcinoma monoclonal antibody ING-1, thehumanized anti-human 17-1A antigen monoclonal antibody 3622W94, thehumanized anti-human colorectal tumor antibody A33, the anti-humanmelanoma antibody directed against GD3 ganglioside R24, or the chimericanti-human squamous-cell carcinoma monoclonal antibody SF-25, ananti-human EGFR antibody, an anti-human EGFRvIII antibody, an anti-humanPSMA antibody, and anti-human PSCA antibody, an anti-human CD22antibody, an anti-human CD30 antibody, an anti-human CD33 antibody, ananti-human CD38 antibody, an anti-human CD40 antibody, an anti-humanCD45 antibody, an anti-human CD52 antibody, an anti-human CD138antibody, an anti-human HLA-DR variant antibody, an anti-human EpCAMantibody, an anti-human CEA antibody, an anti-human MUC1 antibody, ananti-human MUC1 core protein antibody, an anti-human aberrantlyglycosylated MUC1 antibody, an antibody against human fibronectinvariants containing the ED-B domain, and an anti-human HER2/neuantibody.

In another aspect, the claimed invention is directed to a method forproducing a polypeptide in a host cell comprising culturing any of theabove-described the host cells under conditions which permit theproduction of said polypeptide having increased Fc-mediated cellularcytotoxicity. In a preferred embodiment, the method further comprisesisolating said polypeptide having increased Fc-mediated cellularcytotoxicity.

In a further preferred embodiment, the host cell comprises at least onenucleic acid encoding a fusion protein comprising a region equivalent toa glycosylated Fc region of an immunoglobulin.

In a preferred embodiment, the proportion of bisected oligosaccharidesin the Fc region of said polypeptides is greater than 50%, morepreferably, greater than 70%. In another embodiment, the proportion ofbisected hybrid oligosaccharides or galactosylated complexoligosaccharides or mixtures thereof in the Fc region is greater thanthe proportion of bisected complex oligosaccharides in the Fc region ofsaid polypeptide.

In a preferred aspect of the claimed method, the polypeptide is ananti-CD20 antibody and the anti-CD20 antibodies produced by said hostcell have a glycosylation profile, as analyzed by MALDI/TOF-MS, that issubstantially equivalent to that shown in FIG. 2E.

In another preferred aspect of the claimed method, the polypeptide isthe chG250 monoclonal antibody and the chG250 antibodies produced bysaid host cell have a glycosylaton profile, as analyzed by MALDI/TOF-MS,that is substantially equivalent to that shown in FIG. 7D.

In a further aspect, the claimed invention is directed to an antibodyhaving increased antibody dependent cellular cytotoxicity (ADCC)produced by any of the methods described above. In preferredembodiments, the antibody is selected from the group consisting of ananti-CD20 antibody, chCE7, ch-G250, a humanized anti-HER2 monoclonalantibody, ING-1, 3622W94, SF-25, A33, and R24. Alternatively, thepolypeptide can be an antibody fragment that includes a regionequivalent to the Fc region of an immunoglobulin, having increasedFc-mediated cellular cytotoxicity produced by any of the methodsdescribed above.

In a further aspect, the claimed invention is directed to a fusionprotein that includes a region equivalent to the Fc region of animmunoglobulin and having increased Fc-mediated cellular cytotoxicityproduced by any of the methods described above.

In a further aspect, the claimed invention is directed to apharmaceutical composition comprising the antibody, antibody fragment,or fusion protein of the invention and a pharmaceutically acceptablecarrier.

In a further aspect, the claimed invention is directed to a method forthe treatment of cancer comprising administering a therapeuticallyeffective amount of said pharmaceutical composition to a patient in needthereof.

In a further aspect, the invention is directed to an improved method fortreating an autoimmune disease produced in whole or in part bypathogenic autoantibodies based on B-cell depletion comprisingadministering a therapeutically effective amount of immunologicallyactive antibody to a human subject in need thereof, the improvementcomprising administering a therapeutically effective amount of anantibody having increased ADCC prepared as described above. In apreferred embodiment, the antibody is an anti-CD20 antibody. Examples ofautoimmune diseases or disorders include, but are not limited to,immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpurea and chronic idiopathic thrombocytopenicpurpurea, dermatomyositis, Sydenham's chorea, lupus nephritis, rheumaticfever, polyglandular syndromes, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, erythema multiforme, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, primarybiliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, polymyaglia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis,inflammatory responses such as inflammatory skin diseases includingpsoriasis and dermatitis (e.g. atopic dermatitis); systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); respiratory distresssyndrome (including adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; allergic conditions such as eczema and asthma andother conditions involving infiltration of T cells and chronicinflammatory responses; atherosclerosis; leukocyte adhesion deficiency;rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetesmellitus (e.g. Type 1 diabetes mellitus or insulin dependent diabetesmellitus); multiple sclerosis; Reynaud's syndrome; autoimmunethyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenileonset diabetes; and immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes typically foundin tuberculosis, sarcoidosis, polymyositis, granulomatosis andvasculitis; pernicious amenia (Addison's disease); diseases involvingleukocyte diapedesis; central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia (including,but not limited to cryoglobinemia or Coombs positive anemia); myastheniagravis; antigen-antibody complex mediated diseases; anti-glomerularbasement membrane disease; antiphospholipid syndrome; allergic neuritis;Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc. Inthis aspect of the invention, the antibodies of the invention are usedto deplete the blood of normal B-cells for an extended period.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Indirect immunofluorescence assay showing the reactivity of theantibody preparation C2B8-25t to CD20 positive SB cells. Negativecontrols, including the HSB CD20 negative cell line and cells treatedonly with the secondary FITC-conjugated anti-human Fc polyclonalantibody are not shown.

FIG. 2A-2E. MALDI/TOF-MS spectra of the oligosaccharides derived fromMabthera™ (FIG. 2A), C2B8-nt (FIG. 2B), C2B8-2000t (FIG. 2C), C2B8-50t(FIG. 2D), and C2B8-25t (FIG. 2E) antibody samples. Oligosaccharidesappear as [M+Na⁺] and [M+K⁺] ions. Oligosaccharide appearing in thefirst two spectra were derived from cell cultures that do not expressGnTIII, whereas oligosaccharides in FIG. 2C, FIG. 2D, and FIG. 2E werederived from a single cell line expressing GnTIII at different levels(i.e. tetracycline concentrations).

FIGS. 3A and 3B. Illustration of a typical human IgG Fc-associatedoligosaccharide structure (FIG. 3A) and partial N-linked glycosylationpathway (FIG. 3B). (FIG. 3A) The core of the oligosaccharide is composedof three mannose (M) and two N-acetylglucosamine (Gn) monosaccharideresidues attached to Asn₂₉₇. Galactose (G), fucose (F), and bisectingN-acetylglucosamine (Gn, boxed) can be present or absent. TerminalN-acetylneuraminic acid may be also present but it is not included inthe figure. (FIG. 3B) Partial N-linked glycosylation pathway leading tothe formation of the major oligosaccharide classes (dotted frames).Bisecting N-acetylglucosamine is denoted as Gn^(b). Subscript numbersindicate how many monosaccharide residues are present in eacholigosaccharide. Each structure appears together with itssodium-associated [M+Na⁺] mass. The mass of those structures thatcontain fucose (f) are also included.

FIGS. 4A and 4B. ADCC activities of Rituximab glycosylation variants.The percentage of cytotoxicity was measured via lysis of ⁵¹Cr labeledCD20-positive SB cells by human lymphocytes (E:T ratio of 100:1)mediated by different mAb concentrations. (FIG. 4A) Activity of C2B8samples derived from a single cell line but produced at increasingGnTIII expression levels (i.e., decreasing tetracycline concentrations).The samples are C2B8-2000t, C2B8-50t, C2B8-25t, and C2B8-nt (control mAbderived from a clone that does not express GnTIII (FIG. 4B) ADCCactivity of C2B8-50t and C2B8-25t compared to Mabthera™.

FIG. 5. Western blot analysis of the seven GnTIII expressing clones andthe wild type. 30 μg of each sample were loaded on a 8.75% SDS gel,transferred to a PVDF membrane and probed with the anti-c-myc monoclonalantibody (9E10). WT refers to wt-chG250-SP2/0 cells.

FIG. 6. SDS polyacrylamide gel electrophoresis of resolved purifiedantibody samples.

FIG. 7A-7D. MALDI/TOF-MS spectra of neutral oligosaccharide mixturesfrom chG250 mAb samples produced by clones expressing different GnTIIIlevels and wt-chG250-SP2/0 cells: WT (FIG. 7A), 2F1 (FIG. 7B), 3D3 (FIG.7C), 4E6 (FIG. 7D).

FIG. 8A-8D. MALDI/TOF-MS spectra of neutral oligosaccharide mixturesfrom chG250 mAb samples produced by clones expressing different GnTIIIlevels: 4E8, (FIG. 8A); 5G2, (FIG. 8B); 4G3, (FIG. 8C); 5H12, (FIG. 8D).

FIG. 9. In vitro ADCC assay of antibody samples derived from controlwt-chG250-SP2/-cells and GnTIII transected clones 3D3 and 5H12.

DETAILED DESCRIPTION OF THE INVENTION

Terms are used herein as generally used in the art, unless otherwisedefined as follows:

As used herein, the term antibody is intended to include whole antibodymolecules, antibody fragments, or fusion proteins that include a regionequivalent to the Fc region of an immunoglobulin.

As used herein, the term region equivalent to the Fc region of animmunoglobulin is intended to include naturally occurring allelicvariants of the Fc region of an immunoglobulin as well as variantshaving alterations which produce substitutions, additions, or deletionsbut which do not decrease substantially the ability of theimmunoglobulin to mediate antibody dependent cellular cytotoxicity. Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity.(See, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990).

As used herein, the term glycoprotein-modifying glycosyl transferaserefers to β(1,4)-N-acetylglucosaminyltransferase III (GnTIII).

As used herein, the terms engineer, engineered, engineering andglycosylation engineering are considered to include any manipulation ofthe glycosylation pattern of a naturally occurring polypeptide orfragment thereof. Glycosylation engineering includes metabolicengineering of the glycosylation machinery of a cell, including geneticmanipulations of the oligosaccharide synthesis pathways to achievealtered glycosylation of glycoproteins expressed in cells. Furthermore,glycosylation engineering includes the effects of mutations and cellenvironment on glycosylation.

As used herein, the term host cell covers any kind of cellular systemwhich can be engineered to generate modified glycoforms of proteins,protein fragments, or peptides of interest, including antibodies andantibody fragments. Typically, the host cells have been manipulated toexpress optimized levels of GnT III. Host cells include cultured cells,e.g., mammalian cultured cells, such as CHO cells, BHK cells, NS0 cells,SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells,PER.C6 cells or hybridoma cells, yeast cells, and insect cells, to nameonly a few, but also cells comprised within a transgenic animal orcultured tissue.

As used herein, the term Fc-mediated cellular cytotoxicity includesantibody-dependent cellular cytotoxicity and cellular cytotoxicitymediated by a soluble Fc-fusion protein containing a human Fc-region. Itis an immune mechanism leading to the lysis of “antibody-targeted cells”by “human immune effector cells”, wherein:

-   -   The “human immune effector cells” are a population of leukocytes        that display Fc receptors on their surface through which they        bind to the Fc-region of antibodies or of Fc-fusion proteins and        perform effector functions. Such a population may include, but        is not limited to, peripheral blood mononuclear cells (PBMC)        and/or natural killer (NK) cells.    -   The “antibody-targeted cells” are cells bound by the antibodies        or Fc-fusion proteins. The antibodies or Fc fusion-proteins bind        to target cells via the protein part N-terminal to the Fc        region.

As used herein, the term increased Fc-mediated cellular cytotoxicity isdefined as either an increase in the number of “antibody-targeted cells”that are lysed in a given time, at a given concentration of antibody, orof Fc-fusion protein, in the medium surrounding the target cells, by themechanism of Fc-mediated cellular cytotoxicity defined above, and/or areduction in the concentration of antibody, or of Fc-fusion protein, inthe medium surrounding the target cells, required to achieve the lysisof a given number of “antibody-targeted cells”, in a given time, by themechanism of Fc-mediated cellular cytotoxicity. The increase inFc-mediated cellular cytotoxicity is relative to the cellularcytotoxicity mediated by the same antibody, or Fc-fusion protein,produced by the same type of host cells, using the same standardproduction, purification, formulation and storage methods, which areknown to those skilled in the art, but that has not been produced byhost cells engineered to express the glycosyltransferase GnTIII by themethods described herein.

By antibody having increased antibody dependent cellular cytotoxicity(ADCC) is meant an antibody having increased ADCC as determined by anysuitable method known to those of ordinary skill in the art. Oneaccepted in vitro ADCC assay is as follows:

1) the assay uses target cells that are known to express the targetantigen recognized by the antigen-binding region of the antibody;

2) the assay uses human peripheral blood mononuclear cells (PBMCs),isolated from blood of a randomly chosen healthy donor, as effectorcells;

3) the assay is carried out according to following protocol:

-   -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labelled with 100 micro-Curies of ⁵¹Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 10⁵ cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04        ng/ml in cell culture medium and 50 microliters of the resulting        antibody solutions are added to the target cells in the 96-well        microtiter plate, testing in triplicate various antibody        concentrations covering the whole concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labelled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the antibody        solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labelled target cells, receive        50 microliters of RPMI cell culture medium instead of the        antibody solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter;    -   x) the percentage of specific lysis is calculated for each        antibody concentration according to the formula        (ER-MR)/(MR-SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that antibody concentration,        MR is the average radioactivity quantified (see point ix above)        for the MR controls (see point v above), and SR is the average        radioactivity quantified (see point ix above) for the SR        controls (see point vi above);

4) “increased ADCC” is defined as either an increase in the maximumpercentage of specific lysis observed within the antibody concentrationrange tested above, and/or a reduction in the concentration of antibodyrequired to achieve one half of the maximum percentage of specific lysisobserved within the antibody concentration range tested above. Theincrease in ADCC is relative to the ADCC, measured with the above assay,mediated by the same antibody, produced by the same type of host cells,using the same standard production, purification, formulation andstorage methods, which are known to those skilled in the art, but thathas not been produced by host cells engineered to overexpress theglycosyltransferase GnTIII.

As used herein, the term anti-CD20 antibody is intended to mean anantibody which specifically recognizes a cell surface non-glycosylatedphosphoprotein of 35,000 Daltons, typically designated as the human Blymphocyte restricted differentiation antigen Bp35, commonly referred toas CD20.

Identification and Generation of Nucleic Acids Encoding a Protein forwhich Modification of the Glycosylation Pattern is Desired

The present invention provides methods for the generation and use ofhost cell systems for the production of glycoforms of antibodies orantibody fragments or fusion proteins which include antibody fragmentswith increased antibody-dependent cellular cytotoxicity. Identificationof target epitopes and generation of antibodies having potentialtherapeutic value, for which modification of the glycosylation patternis desired, and isolation of their respective coding nucleic acidsequence is within the scope of the invention.

Various procedures known in the art may be used for the production ofantibodies to target epitopes of interest. Such antibodies include butare not limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by an Fab expression library. Suchantibodies may be useful, e.g., as diagnostic or therapeutic agents. Astherapeutic agents, neutralizing antibodies, i.e., those which competefor binding with a ligand, substrate or adapter molecule, are ofespecially preferred interest.

For the production of antibodies, various host animals are immunized byinjection with the target protein of interest including, but not limitedto, rabbits, mice, rats, etc. Various adjuvants may be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, saponin, oil emulsions, keyholelimpet hemocyanin, dinitrophenol, and potentially useful human adjuvantssuch as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to the target of interest may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Kohler and Milstein,Nature 256:495-97 (1975), the human B-cell hybridoma technique (Kosboret al., Immunology Today 4:72 (1983); Cote et al., Proc. Natl. Acad.Sci. U.S.A. 80:2026-30 (1983) and the EBV-hybridoma technique (Cole etal., Monoclonal Antibodies and Cancer Therapy 77-96 (Alan R. Liss, Inc.,1985)). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. U.S.A.81:6851-55 (1984); Neuberger et al., Nature 312:604-08 (1984); Takeda etal., Nature 314:452-54 (1985) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to producesingle chain antibodies having a desired specificity.

Antibody fragments which contain specific binding sites of the targetprotein of interest may be generated by known techniques. For example,such fragments include, but are not limited to, F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed (Huse et al., Science 246:1275-81 (1989) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity to the target protein of interest.

Once an antibody or antibody fragment has been identified for whichmodification in the glycosylation pattern are desired, the codingnucleic acid sequence is identified and isolated using techniques wellknown in the art.

-   -   a. Generation of Cell Lines for the Production of Proteins with        Altered Glycosylation Pattern

The present invention provides host cell expression systems for thegeneration of proteins having modified glycosylation patterns. Inparticular, the present invention provides host cell systems for thegeneration of glycoforms of proteins having an improved therapeuticvalue. Therefore, the invention provides host cell expression systemsselected or engineered to increase the expression of aglycoprotein-modifying glycosyltransferase, namelyβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII). Specifically, suchhost cell expression systems may be engineered to comprise a recombinantnucleic acid molecule encoding GnTIII, operatively linked to aconstitutive or regulated promoter system. Alternatively, host cellexpression systems may be employed that naturally produce, are inducedto produce, and/or are selected to produce GnTIII.

In one specific embodiment, the present invention provides a host cellthat has been engineered to express at least one nucleic acid encodingGnTIII. In one aspect, the host cell is transformed or transfected witha nucleic acid molecule comprising at least one gene encoding GnTIII. Inan alternate aspect, the host cell has been engineered and/or selectedin such way that endogenous GnTIII 15 activated. For example, the hostcell may be selected to carry a mutation triggering expression ofendogenous GnTIII. In one specific embodiment, the host cell is a CHOlec10 mutant. Alternatively, the host cell may be engineered such thatendogenous GnTIII 15 activated. In again another alternative, the hostcell is engineered such that endogenous GnTIII has been activated byinsertion of a constitutive promoter element, a transposon, or aretroviral element into the host cell chromosome.

Generally, any type of cultured cell line can be used as a background toengineer the host cell lines of the present invention. In a preferredembodiment, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myelomacells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridomacells, yeast cells, or insect cells are used as the background cell lineto generate the engineered host cells of the invention.

The invention is contemplated to encompass any engineered host cellsexpressing GnTIII as defined herein.

One or several nucleic acids encoding GnTIII may be expressed under thecontrol of a constitutive promoter or, alternately, a regulatedexpression system. Suitable regulated expression systems include, butare not limited to, a tetracycline-regulated expression system, anecdysone-inducible expression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encoding GnTIII are comprisedwithin the host cell system, some of them may be expressed under thecontrol of a constitutive promoter, while others are expressed under thecontrol of a regulated promoter. The maximal expression level isconsidered to be the highest possible level of stable GnTIII expressionthat does not have a significant adverse effect on cell growth rate, andwill be determined using routine experimentation. Expression levels aredetermined by methods generally known in the art, including Western blotanalysis using a GnTIII specific antibody, Northern blot analysis usinga GnTIII specific nucleic acid probe, or measurement of enzymaticactivity. Alternatively, a lectin may be employed which binds tobiosynthetic products of the GnTIII, for example, E₄-PHA lectin. In afurther alternative, the nucleic acid may be operatively linked to areporter gene; the expression levels of the GnTIII are determined bymeasuring a signal correlated with the expression level of the reportergene. The reporter gene may transcribed together with the nucleicacid(s) encoding said GnTIII as a single mRNA molecule; their respectivecoding sequences may be linked either by an internal ribosome entry site(IRES) or by a cap-independent translation enhancer (CITE). The reportergene may be translated together with at least one nucleic acid encodingsaid GnTIII such that a single polypeptide chain is formed. The nucleicacid encoding the GnTIII may be operatively linked to the reporter geneunder the control of a single promoter, such that the nucleic acidencoding the GnTIII and the reporter gene are transcribed into an RNAmolecule which is alternatively spliced into two separate messenger RNA(mRNA) molecules; one of the resulting mRNAs is translated into saidreporter protein, and the other is translated into said GnTIII.

If several different nucleic acids encoding GnTIII are expressed, theymay be arranged in such way that they are transcribed as one or asseveral mRNA molecules. If they are transcribed as a single mRNAmolecule, their respective coding sequences may be linked either by aninternal ribosome entry site (IRES) or by a cap-independent translationenhancer (CITE). They may be transcribed from a single promoter into anRNA molecule which is alternatively spliced into several separatemessenger RNA (mRNA) molecules, which then are each translated intotheir respective encoded GnTIII.

In other embodiments, the present invention provides host cellexpression systems for the generation of therapeutic antibodies, havingan increased antibody-dependent cellular cytotoxicity, and cells whichdisplay the IgG Fc region on the surface to promote Fc-mediatedcytotoxicity. Generally, the host cell expression systems have beenengineered and/or selected to express nucleic acids encoding theantibody for which the production of altered glycoforms is desired,along with at least one nucleic acid encoding GnTIII. In one embodiment,the host cell system is transfected with at least one gene encodingGnTIII. Typically, the transfected cells are selected to identify andisolate clones that stably express the GnTIII. In another embodiment,the host cell has been selected for expression of endogenous GnTIII. Forexample, cells may be selected carrying mutations which triggerexpression of otherwise silent GnTIII. For example, CHO cells are knownto carry a silent GnT III gene that is active in certain mutants, e.g.,in the mutant Lec10. Furthermore, methods known in the art may be usedto activate silent GnTIII, including the insertion of a regulated orconstitutive promoter, the use of transposons, retroviral elements, etc.Also the use of gene knockout technologies or the use of ribozymemethods may be used to tailor the host cell's GnTIII expression level,and is therefore within the scope of the invention.

Any type of cultured cell line can be used as background to engineer thehost cell lines of the present invention. In a preferred embodiment, CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cell,or insect cells may be used. Typically, such cell lines are engineeredto further comprise at least one transfected nucleic acid encoding awhole antibody molecule, an antibody fragment, or a fusion protein thatincludes a region equivalent to the Fc region of an immunoglobulin. Inan alternative embodiment, a hybridoma cell line expressing a particularantibody of interest is used as background cell line to generate theengineered host cells of the invention.

Typically, at least one nucleic acid in the host cell system encodes GnTIII.

One or several nucleic acids encoding GnTIII may be expressed under thecontrol of a constitutive promoter, or alternately, a regulatedexpression system. Suitable regulated expression systems include, butare not limited to, a tetracycline-regulated expression system, anecdysone-inducible expression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encoding GnTIII are comprisedwithin the host cell system, some of them may be expressed under thecontrol of a constitutive promoter, while others are expressed under thecontrol of a regulated promoter. The maximal expression level isconsidered to be the highest possible level of stable GnTIII expressionthat does not have a significant adverse effect on cell growth rate, andwill be determined using routine experimentation. Expression levels aredetermined by methods generally known in the art, including Western blotanalysis using a GnTIII specific antibody, Northern blot analysis usinga GnTIII specific nucleic acid probe, or measurement of GnTIII enzymaticactivity. Alternatively, a lectin may be employed which binds tobiosynthetic products of GnTIII, for example, E₄-PHA lectin. In afurther alternative, the nucleic acid may be operatively linked to areporter gene; the expression levels of the glycoprotein-modifyingglycosyl transferase are determined by measuring a signal correlatedwith the expression level of the reporter gene. The reporter gene maytranscribed together with the nucleic acid(s) encoding saidglycoprotein-modifying glycosyl transferase as a single mRNA molecule;their respective coding sequences may be linked either by an internalribosome entry site (IRES) or by a cap-independent translation enhancer(CITE). The reporter gene may be translated together with at least onenucleic acid encoding GnTIII such that a single polypeptide chain isformed. The nucleic acid encoding the GnTIII may be operatively linkedto the reporter gene under the control of a single promoter, such thatthe nucleic acid encoding the GnTIII and the reporter gene aretranscribed into an RNA molecule which is alternatively spliced into twoseparate messenger RNA (mRNA) molecules; one of the resulting mRNAs istranslated into said reporter protein, and the other is translated intosaid GnTIII.

If several different nucleic acids encoding a GnTIII are expressed, theymay be arranged in such way that they are transcribed as one or asseveral mRNA molecules. If they are transcribed as single mRNA molecule,their respective coding sequences may be linked either by an internalribosome entry site (IRES) or by a cap-independent translation enhancer(CITE). They may be transcribed from a single promoter into an RNAmolecule which is alternatively spliced into several separate messengerRNA (mRNA) molecules, which then are each translated into theirrespective encoded GnTIII.

i. Expression Systems

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of theprotein of interest and the coding sequence of the GnTIII andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y. (1989).

A variety of host-expression vector systems may be utilized to expressthe coding sequence of the protein of interest and the coding sequenceof the GnTIII. Preferably, mammalian cells are used as host cell systemstransfected with recombinant plasmid DNA or cosmid DNA expressionvectors containing the coding sequence of the protein of interest andthe coding sequence of the GnTIII. Most preferably, CHO cells, BHKcells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, orinsect cells are used as host cell system. In alternate embodiments,other eukaryotic host cell systems may be contemplated, including, yeastcells transformed with recombinant yeast expression vectors containingthe coding sequence of the protein of interest and the coding sequenceof the GnTIII; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the coding sequence ofthe protein of interest and the coding sequence of the GnTIII; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence of the protein of interest andthe coding sequence of the GnTIII; or animal cell systems infected withrecombinant virus expression vectors (e.g., adenovirus, vaccinia virus)including cell lines engineered to contain multiple copies of the DNAencoding the protein of interest and the coding sequence of the GnTIIIeither stably amplified (CHO/dhfr) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines).

For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:3567 (1989); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981)); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)genes. Recently, additional selectable genes have been described, namelytrpB, which allows cells to utilize indole in place of tryptophan; hisD,which allows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)); the glutaminesynthase system; and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.(1987)).

ii. Identification of Transfectants or Transformants that Express theProtein Having a Modified Glycosylation Pattern

The host cells which contain the coding sequence and which express thebiologically active gene products may be identified by at least fourgeneral approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) thepresence or absence of “marker” gene functions; (c) assessing the levelof transcription as measured by the expression of the respective mRNAtranscripts in the host cell; and (d) detection of the gene product asmeasured by immunoassay or by its biological activity.

In the first approach, the presence of the coding sequence of theprotein of interest and the coding sequence of the GnTIII inserted inthe expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the respective coding sequences, respectively, or portionsor derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the coding sequence of the protein of interest and the codingsequence of the GnTIII are inserted within a marker gene sequence of thevector, recombinants containing the respective coding sequences can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the coding sequences under thecontrol of the same or different promoter used to control the expressionof the coding sequences. Expression of the marker in response toinduction or selection indicates expression of the coding sequence ofthe protein of interest and the coding sequence of the GnTIII.

In the third approach, transcriptional activity for the coding region ofthe protein of interest and the coding sequence of the GnTIII can beassessed by hybridization assays. For example, RNA can be isolated andanalyzed by Northern blot using a probe homologous to the codingsequences of the protein of interest and the coding sequence of theGnTIII or particular portions thereof. Alternatively, total nucleicacids of the host cell may be extracted and assayed for hybridization tosuch probes.

In the fourth approach, the expression of the protein products of theprotein of interest and the coding sequence of the GnTIII can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active geneproducts.

b. Generation and Use of Proteins and Protein Fragments Having AlteredGlycosylation Patterns

-   -   i. Generation and Use of Antibodies Having Increased        Antibody-Dependent Cellular Cytotoxicity

In preferred embodiments, the present invention provides glycoforms ofantibodies and antibody fragments having increased antibody-dependentcellular cytotoxicity.

Clinical trials of unconjugated monoclonal antibodies (mAbs) for thetreatment of some types of cancer have recently yielded encouragingresults. Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); Deo etal., Immunology Today 18:127 (1997). A chimeric, unconjugated IgG1 hasbeen approved for low-grade or follicular B-cell non-Hodgkin's lymphomaDillman, Cancer Biother. & Radiopharm. 12:223-25 (1997), while anotherunconjugated mAb, a humanized IgG1 targeting solid breast tumors, hasalso been showing promising results in phase III clinical trials. Deo etal., Immunology Today 18:127 (1997). The antigens of these two mAbs arehighly expressed in their respective tumor cells and the antibodiesmediate potent tumor destruction by effector cells in vitro and in vivo.In contrast, many other unconjugated mAbs with fine tumor specificitiescannot trigger effector functions of sufficient potency to be clinicallyuseful. Frost et al., Cancer 80:317-33 (1997); Surfus et al., J.Immunother. 19:184-91 (1996). For some of these weaker mAbs, adjunctcytokine therapy is currently being tested. Addition of cytokines canstimulate antibody-dependent cellular cytotoxicity (ADCC) by increasingthe activity and number of circulating lymphocytes. Frost et al., Cancer80:317-33 (1997); Surfus et al., J. Immunother. 19:184-91 (1996). ADCC,a lytic attack on antibody-targeted cells, is triggered upon binding ofleukocyte receptors to the constant region (Fc) of antibodies. Deo etal., Immunology Today 18:127 (1997).

A different, but complementary, approach to increase ADCC activity ofunconjugated IgG1 s is to engineer the Fc region of the antibody toincrease its affinity for the lymphocyte receptors (FcγRs). Proteinengineering studies have shown that FcγRs interact with the lower hingeregion of the IgG CH2 domain. Lund et al., J. Immunol. 157:4963-69(1996). However, FcγR binding also requires the presence ofoligosaccharides covalently attached at the conserved Asn 297 in the CH2region. Lund et al., J. Immunol. 157:4963-69 (1996); Wright andMorrison, Trends Biotech. 15:26-31 (1997), suggesting that eitheroligosaccharide and polypeptide both directly contribute to theinteraction site or that the oligosaccharide is required to maintain anactive CH2 polypeptide conformation. Modification of the oligosaccharidestructure can therefore be explored as a means to increase the affinityof the interaction.

An IgG molecule carries two N-linked oligosaccharides in its Fc region,one on each heavy chain. As any glycoprotein, an antibody is produced asa population of glycoforms which share the same polypeptide backbone buthave different oligosaccharides attached to the glycosylation sites. Theoligosaccharides normally found in the Fc region of serum IgG are ofcomplex bi-antennary type (Wormald et al., Biochemistry 36:130-38(1997), with low level of terminal sialic acid and bisectingN-acetylglucosamine (GlcNAc), and a variable degree of terminalgalactosylation and core fucosylation. Some studies suggest that theminimal carbohydrate structure required for FcγR binding lies within theoligosaccharide core. Lund et al., J. Immunol. 157:4963-69 (1996) Theremoval of terminal galactoses results in approximately a two-foldreduction in ADCC activity, indicating a role for these residues in FcγRreceptor binding. Lund et al., J. Immunol. 157:4963-69 (1996)

The mouse- or hamster-derived cell lines used in industry and academiafor production of unconjugated therapeutic mAbs normally attach therequired oligosaccharide determinants to Fc sites. IgGs expressed inthese cell lines lack, however, the bisecting GlcNAc found in lowamounts in serum IgGs. Lifely et al., Glycobiology 318:813-22 (1995). Incontrast, it was recently observed that a rat myeloma-produced,humanized IgG1 (CAMPATH-1H) carried a bisecting GlcNAc in some of itsglycoforms. Lifely et al., Glycobiology 318:813-22 (1995). The ratcell-derived antibody reached a similar in vitro ADCC activity asCAMPATH-1H antibodies produced in standard cell lines, but atsignificantly lower antibody concentrations.

The CAMPATH antigen is normally present at high levels on lymphomacells, and this chimeric mAb has high ADCC activity in the absence of abisecting GlcNAc. Lifely et al., Glycobiology 318:813-22 (1995). In theN-linked glycosylation pathway, a bisecting GlcNAc is added by theenzyme β(1,4)-N-acetylglucosaminyltransferase III (GnT III). Schachter,Biochem. Cell Biol. 64:163-81 (1986).

The present inventors used a single antibody-producing CHO cell line,that was previously engineered to express, in an externally-regulatedfashion, different levels of a cloned GnT III gene. This approachestablished for the first time a rigorous correlation between expressionof GnTIII and the ADCC activity of the modified antibody.

The present inventors previously showed that C2B8 antibody modifiedaccording to the disclosed method had an about sixteen-fold higher ADCCactivity than the standard, unmodified C2B8 antibody produced underidentical cell culture and purification conditions. Briefly, a C2B8antibody sample expressed in CHO-tTA-C2B8 cells that do not have GnT IIIexpression showed a cytotoxic activity of about 31% (at 1 μg/ml antibodyconcentration), measured as in vitro lysis of SB cells (CD20+) by humanlymphocytes. In contrast, C2B8 antibody derived from a CHO cell cultureexpressing GnT III at a basal, largely repressed level showed at 1 μg/mlantibody concentration a 33% increase in ADCC activity against thecontrol at the same antibody concentration. Moreover, increasing theexpression of GnT III produced a large increase of almost 80% in themaximal ADCC activity (at 1 μg/ml antibody concentration) compared tothe control at the same antibody concentration. (See InternationalPublication No. WO 99/54342, the entire contents of which are herebyincorporated by reference.)

Further antibodies of the invention having increased antibody-dependentcellular cytotoxicity include, but are not limited to, anti-humanneuroblastoma monoclonal antibody (chCE7) produced by the methods of theinvention, a chimeric anti-human renal cell carcinoma monoclonalantibody (ch-G250) produced by the methods of the invention, a humanizedanti-HER2 monoclonal antibody (e.g., Trastuzumab (HERCEPTIN)) producedby the methods of the invention, a chimeric anti-human colon, lung, andbreast carcinoma monoclonal antibody (ING-1) produced by the methods ofthe invention, a humanized anti-human 17-1A antigen monoclonal antibody(3622W94) produced by the methods of the invention, a humanizedanti-human colorectal tumor antibody (A33) produced by the methods ofthe invention, an anti-human melanoma antibody (R24) directed againstGD3 ganglioside produced by the methods of the invention, and a chimericanti-human squamous-cell carcinoma monoclonal antibody (SF-25) producedby the methods of the invention, an anti-human small cell lung carcinomamonoclonal antibody (BEC2, ImClone Systems, Merck KgaA) produced by themethods of the invention, an anti-human non-Hodgkin's lymphomamonoclonal antibody (Bexxar (tositumomab, Coulter Pharmaceuticals),Oncolym (Techniclone, Alpha Therapeutic)) produced by the methods of theinvention, an anti-human squamous cell head and neck carcinomamonoclonal antibody (C225, ImClone Systems) prepared by the methods ofthe invention, an anti-human rectal and colon carcinoma monoclonalantibody (Panorex (edrecolomab), Centocor, Glaxo Wellcome) prepared bythe methods of the invention, an anti-human ovarian carcinoma monoclonalantibody (Theragyn, Antisoma) produced by the methods of the invention,an anti-human acute myelogenous leukemia carcinoma monoclonal antibody(SmartM195, Protein Design Labs, Kanebo) produced by the methods of theinvention, an anti-human malignant glioma monoclonal antibody (Cotara,Techniclone, Cambridge Antibody Technology) produced by the methods ofthe invention, an anti-human B cell non-Hodgkins lymphoma monoclonalantibody (IDEC-Y2B8, IDEC Pharmaceuticals) produced by the methods ofthe invention, an anti-human solid tumors monoclonal antibody (CEA-Cide,Immunomedics) produced by the methods of the invention, an anti-humancolorectal carcinoma monoclonal antibody (Iodine 131-MN-14,Immunomedics) produced by the methods of the invention, an anti-humanovary, kidney, breast, and prostate carcinoma monoclonal antibody(MDX-210, Medarex, Novartis) produced by the methods of the invention,an anti-human colorectal and pancreas carcinoma monoclonal antibody(TTMA, Pharmacie & Upjohn) produced by the methods of the invention, ananti-human TAG-72 expressing carcinoma monoclonal antibody (MDX-220,Medarex) produced by the methods of the invention, an anti-humanEGFr-expressing carcinoma monoclonal antibody (MDX-447) produced by themethods of the invention, Anti-VEGF monoclonal antibody (Genentech)produced by the methods of the invention, an anti-human breast, lung,prostate and pancreas carcinoma and malignant melanoma monoclonalantibody (BrevaRex, AltaRex) produced by the methods of the invention,and an anti-human acute myelogenous leukemia monoclonal antibody(Monoclonal Antibody Conjugate, Immunex) produced by the methods of theinvention. In addition, the invention is directed to antibody fragmentand fusion proteins comprising a region that is equivalent to the Fcregion of immunoglobulins.

-   -   ii. Generation and Use of Fusion Proteins Comprising a Region        Equivalent to An Fc Region of an Immunoglobulin that Promote        Fc-Mediated Cytotoxicity

As discussed above, the present invention relates to a method forincreasing the ADCC activity of therapeutic antibodies. This is achievedby engineering the glycosylation pattern of the Fc region of suchantibodies, in particular by maximizing the proportion of antibodymolecules carrying bisected complex oligosaccharides and bisected hybridoligosaccharides N-linked to the conserved glycosylation sites in theirFc regions. This strategy can be applied to increase Fc-mediatedcellular cytotoxicity against undesirable cells mediated by any moleculecarrying a region that is an equivalent to the Fc region of animmunoglobulin, not only by therapeutic antibodies, since the changesintroduced by the engineering of glycosylation affect only the Fc regionand therefore its interactions with the Fc receptors on the surface ofeffector cells involved in the ADCC mechanism. Fc-containing moleculesto which the presently disclosed methods can be applied include, but arenot limited to, (a) soluble fusion proteins made of a targeting proteindomain fused to the N-terminus of an Fc-region (Chamov and Ashkenazi,Trends Biotech. 14: 52 (1996) and (b) plasma membrane-anchored fusionproteins made of a type II transmembrane domain that localizes to theplasma membrane fused to the N-terminus of an Fc region (Stabila, P. F.,Nature Biotech. 16: 1357 (1998)).

In the case of soluble fusion proteins (a) the targeting domain directsbinding of the fusion protein to undesirable cells such as cancer cells,i.e., in an analogous fashion to therapeutic antibodies. The applicationof presently disclosed method to enhance the Fc-mediated cellularcytotoxic activity mediated by these molecules would therefore beidentical to the method applied to therapeutic antibodies.

In the case of membrane-anchored fusion proteins (b) the undesirablecells in the body have to express the gene encoding the fusion protein.This can be achieved either by gene therapy approaches, i.e., bytransfecting the cells in vivo with a plasmid or viral vector thatdirects expression of the fusion protein-encoding gene to undesirablecells, or by implantation in the body of cells genetically engineered toexpress the fusion protein on their surface. The later cells wouldnormally be implanted in the body inside a polymer capsule (encapsulatedcell therapy) where they cannot be destroyed by an Fc-mediated cellularcytotoxicity mechanism. However should the capsule device fail and theescaping cells become undesirable, then they can be eliminated byFc-mediated cellular cytotoxicity. Stabila et al., Nature Biotech. 16:1357 (1998). In this case, the presently disclosed method would beapplied either by incorporating into the gene therapy vector anadditional gene expression cassette directing adequate or maximalexpression levels of GnT III or by engineering the cells to be implantedto express adequate or maximal levels of GnT III. In both cases, the aimof the disclosed method is to increase or maximize the proportion ofsurface-displayed Fc regions carrying bisected complex oligosaccharidesand/or bisected hybrid oligosaccharides.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

EXAMPLE 1 New Versions of the Chimeric Anti-CD20 Antibody IDEC-C2B8Having Enhanced Antibody-Dependent Cellular Cytotoxicity Obtained byGlycosylation Engineering of an IDEC-CEB8 Producing Cell Line

Synthesis of VH and VL Coding Regions of IDEC-C2B8 and Construction ofMammalian Expression Vectors. cDNAs encoding the VH and VL regions ofIDEC-C2B8 antibody were assembled from a set of overlappingsingle-stranded oligonucleotides in a one-step process using PCR(Kobayashi, N., et al., Biotechniques 23:500-503 (1997)). The originalsequence data coding for IDEC-C2B8 VL and VH were obtained from apublished international patent application (International PublicationNumber: WO 94/11026). Assembled VL and VH cDNA fragments were subclonedinto pBluescriptIIKS(+), sequenced and directly joined by ligation tothe human constant light (Igκ) and heavy (IgGl) chain cDNAs,respectively, using unique restriction sites introduced at the variableand constant region junctions without altering the original amino acidresidue sequence (Umana, P., et al., Nat Biotechnol. 17:176-180 (1999);Reff, M. E., et al., Blood 83:435-445 (1994)). Each full-length cDNA wasseparately subcloned into pcDNA3.1(+) (Invitrogen, Leek, TheNetherlands) yielding mammalian expression vectors for chimeric C2B8light (pC2B8L) and heavy (pC2B8H) chains.

Production of IDEC-C2B8 in CHO Cells Expressing Different Levels ofGnTIII. Establishment of two CHO cell lines, CHO-tet-GnTIII expressingdifferent levels of GnTIII depending on the tetracycline concentrationin the culture medium; and CHO-tTA, the parental cell line that does notexpress GnTIII has been described previously (Umana, P., et al., NatBiotechnol. 17:176-180 (1999); Umana, P., et al., Biotechnol Bioeng.65:542-549 (1999)). Each cell line was cotranfected with vectors pC2B8L,pC2B8H, and pZeoSV2(+) (for Zeocin resistance; Invitrogen, Leek, TheNetherlands) using a calcium phosphate method. Zeocin resistant cloneswere transferred to a 96-well plate and assayed for IDEC-C2B8 productionusing an ELISA assay specific for the human constant region (4). ThreeIDEC-C2B8 samples were obtained from parallel cultures of a selectedclone (CHO-tet-GnTIII-C2B8), differing only in the tetracyclineconcentration added to the medium (25, 50 and 2000 ng/mL respectively).Culture supernatants were harvested in the late exponential phase. Anadditional antibody sample was obtained from a CHO-tTA-derived clone,CHO-tTA-C2B8, cultured under identical conditions but without addingtetracycline to the medium. Antibody samples were purified from culturemedium by protein A affinity chromatography and buffer exchanged to PBSon a cation exchange column as previously described (Umana, P., et al.,Nat Biotechnol. 17:176-180 (1999)). Antibody concentration was measuredusing a fluorescence-based kit from Molecular Probes (Leiden, TheNetherlands) with Rituximab used as standard.

Indirect Immunofluorescence. CD20-positive cells (SB cells; ATCC depositno. ATCC CCL120) and CD20-negative cells (HSB cells; ATCC deposit no.ATCC CCL120.1) were each incubated for 1 h with 2.5 μg/ml ofCHO-tet-GnTIII-derived IDEC-C2B8 antibody in Hank's balanced saltsolution (GibcoBRL, Basel, Switzerland) and 2% bovine serum albuminfraction V (Roche Diagnostics, Rotkreuz, Switzerland) (HBSSB). As anegative control HBSSB was used instead of C2B8 antibody. AFITC-conjugated, anti-human Fc polyclonal antibody was used as asecondary antibody (SIGMA, St. Louis) for all samples. Cells wereexamined using a Leica fluorescence microscope (Wetzlar, Germany).

Oligosaccharide Profiling by MALDI/TOF-MS. Neutral, N-linkedoligosaccharides were derived from C2B8 antibody samples, MabThera™(European counterpart of Rituximab; kind gift from R. Stahel,Universitåtspital, Switzerland), C2B8-25t, C2B8-50t, C2B8-2000t, andC2B8-nt, (100 μg each) as previously described (Umana, P., et al., NatBiotechnol. 17:176-180 (1999)). Briefly, the antibody samples were firsttreated with Arthrobacter ureafaciens sialidase (Oxford Glycosciences,Abingson, UK) to remove any sialic acid monosaccharide residues. NeutralN-linked oligosaccharides were then released from the desialylatedantibody samples using peptide-N-glycosidase F (Oxford Glycosciences),purified using micro-columns, and analyzed by MALDI/TOF-MS in an EliteVoyager 400 spectrometer (Perseptive Biosystems, Farmingham, Mass.).

ADCC Activity Assay.

Peripheral blood mononuclear cells (PBMC) were separated fromheparinated fresh human blood (in all experiments obtained from the samehealthy donor) by centrifugation over a Ficoll-Paque (Pharmacia Biotech,Dübendorf, Switzerland) gradient. PBMC (effector) were depleted ofmonocytes by plastic adherence. CD20-positive SB (target) cells, werelabeled for 90 min with 100 μCi ⁵¹Cr (Amersham, Dübendorf, Switzerland)at 37° C., washed twice in RPMI (GibcoBRL, Basel, Switzerland) andresuspended at a concentration of 10⁵ cells/ml. Fifty microliters ofC2B8 mAb diluted in RPMI medium was added to 100 μl SB cells (10,000cells/well) in a 96-well round bottom microtiter plate (Greiner,Langenthal, Switzerland), centrifuged at 50×g for 1 min, and incubatedfor 1 h at 4° C. Subsequently, 50 μl of effector cell (suspended at2×10⁷ cells/ml in RPMI medium) were added to each 96-well yielding afinal E:T ratio of 100. Plates were incubated for 4 h at 37° C. and 5%CO₂, supernatant was harvested with a Skatron harvesting system (SkatronInstruments, Sterling, Va.) and counted (ER, experimental release) in aCobra 05005 γ counter (Canberra Packard, Meriden, Conn.). Maximum (MR)and spontaneous (SR) releases were obtained by adding, instead of C2B8mAb, 100 μl of 1% Nonidet (Sigma, St. Louis) or 100 μl of RPMI medium,respectively, to 100 μl labeled target cells. All data points wereperformed in triplicate. Specific lysis (%) was calculated with thefollowing formula: (ER−SR)/(MR−SR)×100.

Results and Discussion

Production of IDEC-C2B8 and Verification of Specific Antigen Binding.CHO-tet-GnTIII cells, with stable, tetracycline-regulated expression ofGnTIII and stable, constitutive expression of IDEC-C2B8, wereestablished and scaled-up for production of a set of antibody samples.During scale-up, parallel cultures from the same clone were grown underthree different tetracycline concentrations, 25, 50 and 2000 ng/ml.These levels of tetracycline had previously been shown to result indifferent levels of GnTIII and bisected oligosaccharides (Umana, P., etal., Nat Biotechnol. 17:176-180 (1999); Umana, P., et al., BiotechnolBioeng. 65:542-549 (1999)). A C2B8-producing, control cell line thatdoes not express GnTIII was also established and cultured under the sameconditions as for the three parallel cultures of CHO-tet-GnTIII. AfterProtein A-affinity chromatography, mAb purity was estimated to be higherthan 95% by SDS-PAGE and Coomassie-blue staining. The samples were namedaccording to the tetracycline concentration added to the culture mediumfor their production: C2B8-25t, C2B8-50t, C2B8-2000t and C2B8-nt (i.e.,no tetracycline for the non-bisected control). Sample C2B8-25t showedspecific antigen binding by indirect immunofluorescence usingCD20-positive and CD20-negative cells (FIG. 1), indicating that thesynthesized VL and VH gene fragments were functionally correct.

Oligosaccharide Profiling with MALDI/TOF-MS. The glycosylation profileof each antibody sample was analyzed by MALDI/TOF-MS of the released,neutral oligosaccharide mix. In this technique, oligosaccharides ofdifferent mass appear as separate peaks in the spectrum and theirproportions are quantitatively reflected by the relative peak heights(Harvey, D. J., Rapid Common Mass Spectrom. 7:614-619 (1993); Harvey, D.J., et al., Glycoconj J. 15:333-338 (1998)). Oligosaccharide structureswere assigned to different peaks based on their expected molecularmasses, previous structural data for oligosaccharides derived from IgGImAbs produced in the same host, and information on the N-linkedoligosaccharide biosynthetic pathway.

A clear correlation was found between GnTIII expression levels (i.e.,tetracycline concentration) and the amount of bisected oligosaccharidesderived from the different antibody samples. As expected, MabThera™ andC2B8-nt, which are derived from hosts that do not express GnTIII, didnot carry bisected oligosaccharides (FIG. 2A and FIG. 2B). In contrast,bisected structures amounted up to approximately 35% of theoligosaccharides pool in sample C2B8-2000t, i.e, at a basal level ofGnTIII expression. In this case, the main bisected oligosaccharide peakswere of complex type, unequivocally assigned to peaks at m/z 1689 andm/z 1851 (FIG. 2C). The next higher GnTIII expression level, sampleC2B8-50t, led to an increase in these peaks (including their associatedpotassium aducts at m/z 1705 and 1861) of around 20%. This increase wasaccompanied by a concomitant reduction of their non-bisectedcounterparts at m/z 1486 and 1648, respectively (FIG. 2D). At thehighest GnTIII expression level, sample C2B8-25t, the main substrate forGnTIII, m/z 1486, decreased to almost base-line level, while complexbisected structures (m/z 1689 and 1851) decreased in favor of increasesin peaks at m/z 1664, 1810 and 1826 (FIG. 2E). These peaks can beassigned either to bisected hybrid compounds, to galactosylated complexoligosaccharides, or to a mixture of both. Their relative increase,however, is consistent with the accumulation of bisected hybridcompounds, as GnTIII overexpression can divert the biosynthetic flux atearly stages of the pathway (see FIG. 3A and FIG. 3B). The amount ofbisected oligosaccharide structures (complex and hybrid type) reachedapproximately 80% for this sample.

ADCC Activity of IDEC-C2B8 Glycosylated Variants. Different C2B8 mAbglycosylationvariants were compared for ADCC activity, measured as invitro lysis of CD20-positive SB cells. An additional mAb sample,C2B8-nt, derived from the parental cell line lacking GnTIII, was alsostudied. Sample C2B8-2000t produced at the basal GnTIII expression leveland carrying low levels of bisected oligosaccharides was slightly moreactive than C2B8-nt (FIG. 4A). At the next higher level ofGnTIII-expression, sample C2B8-50t carried approximately equal levels ofbisected and non-bisected oligosaccharides, but did not mediatesignificantly higher target-cell lysis. However, at the lowesttetracycline concentration, sample C2B8-25t, which contained up to 80%of bisected oligosaccharide structures, was significantly more activethan the rest of the samples in the whole antibody concentration range.It reached the maximal level of ADCC activity of sample C2B8-nt at a10-fold lower antibody concentration (FIG. 4A). Sample C2B8-25t alsoshowed a significant increase in the maximal ADCC activity with respectto the control (50% vs. 30% lysis).

Samples C2B8-50t and C2B8-25t, bearing the highest proportions ofbisected oligosaccharides, were further compared in ADCC activity toMabthera™, the version of Rituxan™ currently marketed in Europe (FIG.4B). Sample C2B8-50t showed a slight increase in activity whereas sampleC2B8-25t clearly outperformed Mabthera™ at all antibody concentrations.Approximately a five to ten-fold lower concentration of C2B8-25t wasrequired to reach the maximal ADCC activity of Mabthera™, and themaximal activity of C2B8-25t was about 25% higher than that ofMabthera™.

These results show that, in general, the in vitro ADCC activity of theC2B8 antibody correlates with the proportion of molecules carryingbisected oligosaccharides in the Fc region. We had previously reportedthat in the case of chCE7, an antibody with a low baseline level of ADCCactivity, significant increases of activity could be obtained byincreasing the fraction of bisected oligosaccharides above the levelsfound in naturally-occurring antibodies (Umana, P., et al., NatBiotechnol. 17:176-180 (1999)). The same is true for the C2B8 mAb, whichalready has high ADCC activity in the absence of bisectedoligosaccharides. In the case of chCE7, however, very large increases ofADCC activity were observed at a level of GnTIII expression wherebisected oligosaccharides were predominantly of complex type (Umana, P.,et al., Nat Biotechnol. 17:176-180 (1999)). For the potent C2B8 mAb,such a large boost in activity was only observed at the highest levelsof GnTIII expression studied, where bisected oligosaccharides hadshifted mainly to the hybrid type (FIG. 2). For both mAbs, the sampleswith the highest activities had considerably higher levels of bisectedthan non-bisected oligosaccharides. Together, these observationsindicate that probably both complex and hybrid bisected oligosaccharidesare important for ADCC activity.

In both complex and hybrid oligosaccharides, a bisecting GlcNAc leads toa large change in oligosaccharide conformations (Balaji, P. V., et al.,Int. J. Biol. Macromol. 18:101-114 (1996)). The change occurs in a partof the oligosaccharide that interacts extensively with the polypeptidein the CH2 domain (Jefferis, R., et al., Immunol Rev. 163:59-76 (1998)).Since the polypeptide is relatively flexible at this location (Jefferis,R., et al., Immunol Rev. 163:59-76 (1998)), it is possible that thebisecting N-acetylglucosamine is mediating its biological effectsthrough a conformational change in the Fc region. The potentiallyaltered conformations would already exist in nature, as all serum IgGscarry bisected oligosaccharides. The main difference between theengineered and natural antibodies would be the proportion of moleculesdisplaying the more active conformations.

Various approaches for increasing the activity of unconjugated mAbs arecurrently under clinical evaluation, including radio-immunotherapy,antibody-dependent enzyme/prodrugtherapy, immunotoxins and adjuvanttherapy with cytokines (Hjelm Skog, A., et al., Cancer ImmunolImmunother. 48:463-470 (1999); Blakey, D. C., et al., Cell Biophys.25:175-183 (1994); Wiseman, G. A., et al., Clin Cancer Res.5:3281s-3296s (1999); Hank, J. A., et al., Cancer Res. 50:5234-5239(1990)). These technologies can give large increases in activity, butthey can also lead to significantly higher side effects, elevatedproduction costs and complex logistics from production to administrationto patients when compared to unconjugated mAbs. The technology presentedhere offers an alternative way to obtain increases in potency whilemaintaining a simple production process, and should be applicable tomany unconjugated mAbs.

EXAMPLE 2 New Versions of the Anti-Renal Cell Carcinoma Antibody chG250Having Enhanced Antibody-Dependent Cellular Cytotoxicity Obtained byGlycosylation Engineering of a chG250 Producing Cell Line

1. Cell Culture

SP2/0 mouse myeloma cells producing chG250 chimeric mAb (wt-chG250-SP2/0cells) were grown in standard cell culture medium supplemented with1:100 (v/v) penicillin/streptomycin/antimycotic solution (SIGMA, Buchs,Switzerland). Cells were cultured at 37° C. in a 5% CO₂ humidifiedatmosphere in Tissue Culture Flasks. Medium was changed each 3-4 days.Cells were frozen in culture medium containing 10% DMSO.

2. Generation of SP2/0 Cells with pGnTIII-Puro Expression

wt-chG250-SP2/0 myeloma cells were transfected by electroporation with avector for constitutive expression of GnTIII operatively linked via anIRES to a puromycin resistance gene. 24 hours before electroporationculture medium was changed and cells were seeded at 5×10⁵ cells/ml.Seven million cells were centrifuged for 4 min at 1300 rpm at 4° C.Cells were washed with 3 mL new medium and centrifuged again. Cells wereresuspended in a volume of 0.3-0.5 ml of reaction mix, containing 1.25%(v/v) DMSO and 20-30 μg DNA in culture medium. The electroporation mixwas then transferred to a 0.4 cm cuvette and pulsed at low voltage(250-300 V) and high capacitance (960 μF) using Gene Pulser from BioRad. After electroporation cells were quickly transferred to 6 mL 1.25%(v/v) DMSO culture medium in a T25 culture flask and incubated at 37° C.Stable integrants were selected by applying 2 μg/mL puromycin to themedium two days after electroporation. After 2-3 weeks a stable,puromycin-resistant mixed population was obtained. Single-cell derivedclones were obtained via FACS and were subsequently expanded andmaintained under puromycin selection.

3. Western Blot

Puromycin-resistant clones were screened for GnTIII expression byWestern blotting. The Western blots clearly showed that clones 5H12, 4E6and 4E8 were expressing the highest levels of GnTIII. 5G2 also showed aGnTIII band of middle intensity, whereas 2F1, 3D3 and 4G3 had the lowestband intensities, therefore expressing lower amounts of GnTIII (FIG. 5).

4. Production and Purification of chG250 Monoclonal Antibody from SevenGnTIII-expressing clones including wild type

Clones 2F1, 3D3, 4E6, 4E8, 4G3, 5G2, 5H12 and the wild type(wt-chG250-SP2/0 cells) were seeded at 3×10⁵ cells/mL in a total volumeof 130 ml culture medium, and cultivated in single Triple-flasks. Cellsused for seeding were all in full exponential growth phase, thereforecells were considered to be at the same growth state when the productionbatches started. Cells were cultivated for 4 days. Supernatantscontaining the antibody were collected in the late exponential growthphase to ensure reproducibility. The chG250 monoclonal antibody waspurified in two chromatographic steps. Culture supernatants containingthe chG250 monoclonal antibody derived from each batch were firstpurified using a HiTrap Protein A affinity chromatography. Protein A ishighly specific for the human IgG F_(c) region. Pooled samples from theprotein A eluate were buffer exchanged to PBS by cation-exchangechromatography on a Resource S 1 ml column (Amersham Pharmacia Biotech).Final purity was judged to be higher than 95% from SDS-staining andCoomassie blue staining (FIG. 6). The concentration of each sample wasdetermined with a standard calibration curve using wild type antibodywith known concentration.

5. Oligosaccharide Profiling of mAb Preparations Derived from the SevenClones Expressing Different GnTIII Levels

Oligosaccharide profiles were obtained by matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI/TOF-MS),which accurately provides the molecular masses of the differentoligosaccharide structures. This technique allows a quantitativeanalysis of proportions between different oligosaccharide structureswithin a mixture. Neutral oligosaccharides appeared predominantly as[M+Na⁺] ions, however sometimes they were accompanied by smaller [M+K⁺]ions, leading to an increase in mass of m/z of 16. The percentage of thestructure appearing as potassium ion adducts depends on the content ofthe matrix and may thus vary between samples. A mixture of neutralN-linked oligosaccharides derived from each antibody preparation wasanalyzed using a 2,5-dehydrobenzoic acid (2,5-DHB) as matrix. Some ofthe peaks in the spectra were unequivocally assigned to specificoligosaccharide structures, because of known monosaccharide compositionand unique mass. However, sometimes multiple structures could beassigned to a particular mass. MALDI enables the determination of themass and cannot distinguish between isomers. Knowledge of thebiosynthetic pathway and previous structural data enable, in most cases,the assignment of an oligosaccharide structure to a peak in thespectrum.

Oligosaccharides derived from the mAb sample produced in wt-chG250-SP2/0cell line, that does not express GnTIII, contained nonbisectedbiantennary complex (m/z 1486) and mono- or di-galactosylatednonbisected biantennary complex structures (FIG. 7A), bothα(1,6)-fucosylated in the core region (peaks m/z 1648 and 1810respectively).

Expression of GnTIII generated bisected F_(c)-associated oligosaccharidestructures of two types: complex or hybrid. Complex bisectedoligosaccharides were unequivocally assigned to peaks at m/z 1543, 1689,1705, 1851 and 1867 ([M+K⁺] adduct). As expected, the increase inbisected oligosaccharides was accompanied by a concomitant reduction ofpeaks m/z 1486 and 1648, that correspond to nonbisected complexoligosaccharides. For all samples derived from the GnTIII expressingclones, the main substrate of GnTIII (m/z 1486) decreased dramatically.As expected, the percentage of the nonbisected complex oligosaccharidetype, assigned to peak at m/z 1648, had the lowest values for the clonesexpressing the highest GnTIII levels (clones 4E6, 4E8, 5G2 and 5H12).These two peaks decreased in favor of the accumulation of bisectedcomplex and bisected hybrid type oligosaccharides (FIGS. 7A-7D and8A-8D). The percentage of bisected complex oligosaccharides was higherfor the samples derived from the clones expressing lower amounts ofGnTIII. This is consistent with the fact that a higher GnTIII expressionlevel probably shifts the biosynthetic flux to bisected hybridstructures, thereby decreasing the relative proportions of complex andcomplex bisected compound. For bisected hybrid structures, two possiblestructures could sometimes be assigned to a single peak. Therefore, someassumptions were made in order to approximate the percentage of thesestructures over the total oligosaccharide pool. Peaks m/z 1664, 1680,1810 and 1826 can be assigned to either bisected hybrid type, togalactosylated complex oligosaccharides, or a mixture of them. Due tothe fact that the wt-antibody preparation had a relatively lowpercentage of peak 1664, it was assumed that this peak, appearing insignificant amounts in the antibody samples derived from the differentclones, corresponded entirely to bisected hybrid structures (FIGS. 7A-7Dand 8A-8D). However to assign a specific structure to peaks m/z 1810 and1826 further characterization has to be performed. In summary, byoverexpression of GnTIII, bisected oligosaccharides structures weregenerated and their relative proportions correlated with GnTIIIexpression levels.

6. Measurement of Antibody Mediated Cytotoxic Activity by Calcein-AMRetention

The Calcein-AM retention method of measuring cytotoxicity measures thedye fluorescence remaining in the cells after incubation with theantibody. Four million G250 antigen-positive cells (target) werelabelled with 10 μM Calcein-AM (Molecular Probes, Eugene, Oreg.) in 1.8mL RPMI-1640 cell culture medium (GIBCO BRL, Basel, Switzerland)supplemented with 10% fetal calf serum for 30 min at 37° C. in a 5% CO₂humidified atmosphere. The cells were washed twice in culture medium andresuspended in 12 mL AIMV serum free medium (GIBCO BRL, Basel,Switzerland). Labelled cells were then transferred to U-bottom 96-wells(30,000 cells/well) and incubated in triplicate with differentconcentrations of antibody for 1 hour at 4° C. Peripheral bloodmononuclear cells (PBMC) were separated from heparinated fresh humanblood (in all experiments obtained from the same healthy donor) bycentrifugation over a Ficoll-Paque (Pharmacia Biotech, Dübendorf,Switzerland) gradient. PBMCs were added in triplicate wells in a 50 μLvolume, yielding an effector to target ratio (E:T ratio) of 25:1 and afinal volume of 200 μL. The 96-well plate was then incubated for 4 hoursat 37° C. in a 5% CO₂ atmosphere. Thereafter the 96-well plate wascentrifuged at 700×g for 5 min and the supernatants were discarded. Thecell pellets were washed twice with Hank's balanced salt solution (HBSS)and lysed in 200 μL 0.05M sodium borate, pH 9, 0.1% Triton X-100.Retention of the fluorescent dye in the target cells was measured with aFLUOstar microplate reader (BMG Lab Technologies, Offenburg, Germany).The specific lysis was calculated relative to a total lysis control,resulting from exposure of the target cells to saponin (200 μg/mL inAIMV; SIGMA, Buchs, Switzerland) instead of exposure to antibody.Specific lysis (%) was calculated with the following formula:

${\%\mspace{14mu}{Cytotoxicity}} = \frac{F_{med} - F_{\exp}}{F_{med} - F_{\det}}$where F_(med) represents the fluorescence of target cells treated withmedium alone and considers unspecific lysis by PMBCs, F_(exp) representsthe fluorescence of cells treated with antibody and F_(det) representsthe fluorescence of cells treated with saponin instead of antibody.

To determine the effect of modified glycosylation variants of chG250 onthe in vitro ADCC activity, G250 antigen-positive target cells werecultured with PBMCs with and without chG250 antibody samples atdifferent concentrations. The cytotoxicity of unmodified chG250 antibodyderived from the wild type cell line was compared with two antibodypreparations derived from two cell lines (3D3, 5H12) expressingintermediate and high GnTIII levels, respectively (see FIG. 5).

Unmodified chG250 antibody did not mediate significant ADCC activityover the entire concentration range used in the assay (the activity wasnot significantly different from background). Augmented ADCC activity(close to 20%, see FIG. 9) at 2 μg/mL was observed with the antibodysample derived from clone 3D3, which expressed intermediate GnTIIIlevels. The cytotoxic activity of this antibody samples did not grow athigher antibody concentrations. As expected the antibody preparationderived from clone 5H12 showed a striking increase over samples 3D3 andunmodified antibody in its ability to mediate ADCC against target cells.The maximal ADCC activity of this antibody preparation was around 50%and was remarkable in mediating significant ADCC activity at 125-foldless concentrated when comparing with the unmodified control sample.

EXAMPLE 3 Treatment of Immune-Mediated Thrombocytopenia in a Patientwith Chronic Graft-Versus-Host Disease

Autoimmune thrombocytopenia in chronic graft-versus-host diseaserepresents an instance of B-cell dysregulation leading to clinicaldisease. To treat immune-mediated thrombocytopenia in a subject withchronic graft-versus-host disease, an anti-CD20 chimeric monoclonalantibody prepared by the methods of the present invention and havingincreased ADCC is administered to the subject as described inRatanatharathorn, V. et al., Ann. Intern. Med. 133(4):275-79 (2000) (theentire contents of which is hereby incorporated by reference).Specifically, a weekly infusion of the antibody, 375 mg/m² isadministered to the subject for 4 weeks. The antibody therapy produces amarked depletion of B cells in the peripheral blood and decreased levelsof platelet-associated antibody.

EXAMPLE 4 Treatment of Severe, Immune-Mediated, Pure Red Cell Aplasiaand Hemolytic Anemia

Immune-mediated, acquired pure red cell aplasia (PRCA) is a raredisorder frequently associated with other autoimmune phenomena. To treatimmune-mediated, acquired pure red cell aplasia in a subject, ananti-CD20 chimeric monoclonal antibody prepared by the methods of thepresent invention and having increased ADCC is administered to thesubject as described in Zecca, M. et al., Blood 12:3995-97 (1997) (theentire contents of which are hereby incorporated by reference).Specifically, a subject with PRCA and autoimmune hemolytic anemia isgiven two doses of antibody, 375 mg/m², per week. After antibodytherapy, substitutive treatment with intravenous immunoglobulin isinitiated. This treatment produces a marked depletion of B cells and asignificant rise in reticulocyte count accompanied by increasedhemoglobin levels.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

What is claimed is:
 1. A glycoengineered antigen binding moleculeproduced by a method comprising: (a) culturing a host cell underconditions which permit the production of the glycoengineered antigenbinding molecule, and (b) isolating the glycoengineered antigen bindingmolecule; wherein the glycoengineered antigen binding molecule comprisesan immunoglobulin G (IgG) Fc region containing N-linked oligosaccharidesand has increased Fc-mediated cellular cytotoxicity compared to acorresponding antigen binding molecule that has not beenglycoengineered, and wherein the host cell is engineered to express atleast one nucleic acid encoding β(1,4)-N-acetylglucosaminyltransferaseIII (GnT III) in an amount sufficient to increase the ratio of GlcNAcresidues to fucose residues in the IgG Fc region compared to acorresponding antigen binding molecule that has not beenglycoengineered.
 2. The glycoengineered antigen binding molecule ofclaim 1, wherein the glycoengineered antigen binding molecule is anantibody.
 3. The glycoengineered antigen binding molecule of claim 2,wherein the antibody is selected from the group consisting of: chCE7, ahumanized anti-HER2 monoclonal antibody, ING-1,3622W94, SF-25, A33, andR24.
 4. A pharmaceutical composition comprising the antibody of claim 2and a pharmaceutically acceptable carrier.
 5. The glycoengineeredantigen binding molecule of claim 2, wherein the antibody is IgG.
 6. Apharmaceutical composition comprising the antibody fragment of claim 5and a pharmaceutically acceptable carrier.
 7. The glycoengineeredantigen binding molecule of claim 5, wherein the antibody is IgGl.
 8. Apharmaceutical composition comprising the fusion protein of claim 7 anda pharmaceutically acceptable carrier.
 9. The glycoengineered antigenbinding molecule of claim 2, wherein the antibody is a monoclonalantibody.
 10. The glycoengineered antigen binding molecule of claim 9,wherein the monoclonal antibody is an anti-CD20 monoclonal antibody. 11.The glycoengineered antigen binding molecule of claim 10, wherein theantibody is IDEC-C2B8.
 12. The glycoengineered antigen binding moleculeof claim 11, wherein the antibody has a glycosylation profile, asanalyzed by MALDI/TOF-MS, that is substantially equivalent to that shownin FIG. 2E.
 13. The glycoengineered antigen binding molecule of claim 2,wherein the antibody is a chimeric antibody.
 14. The glycoengineeredantigen binding molecule of claim 13, wherein the chimeric antibody isan anti-CD20 chimeric antibody.
 15. The glycoengineered antigen bindingmolecule of claim 14, wherein the chimeric antibody is chG250.
 16. Theglycoengineered antigen binding molecule of claim 15, wherein theantibody has a glycosylation profile, as analyzed by MALDI/TOF-MS, thatis substantially equivalent to that shown in FIG. 7D.
 17. Theglycoengineered antigen binding molecule of claim 2, wherein theantibody is a humanized antibody.
 18. The glycoengineered antigenbinding molecule of claim 17, wherein the humanized antibody is ananti-CD20 humanized antibody.
 19. The glycoengineered antigen bindingmolecule of claim 1, wherein the glycoengineered antigen bindingmolecule is an antibody fragment.
 20. The glycoengineered antigenbinding molecule of claim 1, wherein the glycoengineered antigen bindingmolecule is a fusion protein.
 21. The glycoengineered antigen bindingmolecule of claim 1, wherein the N-linked oligosaccharides in the IgG Fcregion of the glycoengineered antigen binding molecule are bisectedoligosaccharides.
 22. The glycoengineered antigen binding molecule ofclaim 21, wherein the N-linked oligosaccharides in the IgG Fc region ofthe glycoengineered antigen binding molecule are bisected hybridoligosaccharides or galactosylated complex oligosaccharides or a mixturethereof.
 23. The glycoengineered antigen binding molecule of claim 1,wherein the glycoengineered antigen binding molecule has an increasedproportion of bisecting GlcNAc residues in the IgG Fc region as comparedto a corresponding antigen binding molecule that has not beenglycoengineered.
 24. The glycoengineered antigen binding molecule ofclaim 23, wherein the N-linked oligosaccharides in the IgG Fc region ofthe glycoengineered antigen binding molecule are not high-mannosestructures.
 25. The glycoengineered antigen binding molecule of claim 1,wherein the N-linked oligosaccharides in the IgG Fc region of theglycoengineered antigen binding molecule are nonfucosylatedoligosaccharides.
 26. The glycoengineered antigen binding molecule ofclaim 1, wherein the glycoengineered antigen binding molecule has anincreased ratio of GlcNAc residues to fucose residues in the IgG Fcregion compared to a corresponding antigen binding molecule that has notbeen glycoengineered.
 27. The glycoengineered antigen binding moleculeof claim 1, wherein the increased Fc-mediated cellular cytotoxicity isincreased antibody dependent cellular cytotoxicity (ADCC).
 28. Theglycoengineered antigen binding molecule of claim 27, wherein theglycoengineered antigen binding molecule exhibits at least an 80%increase in maximal ADCC activity compared to a corresponding antigenbinding molecule that has not been glycoengineered.
 29. Aglycoengineered antigen binding molecule produced by a methodcomprising: (a) culturing a mammalian host cell under conditions whichpermit the production of the glycoengineered antigen binding molecule,and (b) isolating the glycoengineered antigen binding molecule; whereinthe glycoengineered antigen binding molecule comprises an IgG Fc regioncontaining nonfucosylated N-linked oligosaccharides and has increasedFc-mediated cellular cytotoxicity compared to a corresponding antigenbinding molecule that has not been glycoengineered, wherein themammalian host cell is engineered to express at least one nucleic acidencoding mammalian GnT III in an amount sufficient to increase the ratioof GlcNAc residues to fucose residues in the IgG Fc region compared to acorresponding antigen binding molecule that has not beenglycoengineered.
 30. A glycoengineered antigen binding molecule producedby a method comprising: (a) culturing a human host cell under conditionswhich permit the production of the glycoengineered antigen bindingmolecule, and (b) isolating the glycoengineered antigen bindingmolecule; wherein the glycoengineered antigen binding molecule comprisesan IgG Fc region containing nonfucosylated N-linked oligosaccharides andhas increased Fc-mediated cellular cytotoxicity compared to acorresponding antigen binding molecule that has not beenglycoengineered, and wherein the human host cell is engineered toexpress at least one nucleic acid encoding human GnT III in an amountsufficient to increase the ratio of GlcNAc residues to fucose residuesin the IgG Fc region compared to a corresponding antigen bindingmolecule that has not been glycoengineered.